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A DNA Barcoding Reference Database for Priority Southern African Snakes

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A DNA Barcoding Reference Database for Priority Southern African Snakes 581 A DNA barcoding reference database for priority southern African snakes 1,2Mimmie M. Kgaditse, 1M. Mwale, and 1A. Kotze 1National Zoological Gardens of South Africa, PO BOX 754, Pretoria 0001, South Africa. 2University of the Witwatersrand, APES, P/Bag 3, Johannesburg, South Africa ABSTRACT Anthropogenic impacts on the environment have led to drastic effects on community assemblies and the diversity of species. Given their ectothermic physiological characteristics and specific niche requirements, several southern African snakes are particularly vulnerable to these effects. There is also a need for monitoring the legal trade of snake species to ensure that only non-regulated species are collected from the wild. The aim of this study was to set up a DNA barcoding species reference database of priority species that are currently listed as threatened or endangered on CITES appendices and IUCN Red lists and also included on permit requests for the pet trade. Look-alikes species whose specimens have similar features and can be confused with priority species that are illegal to trade for conservation reasons were also included. APPROACH The priority and look-alike species To demonstrate the utility and effectiveness of DNA barcoding for species PRIORITY SPECIES LOOK-ALIKES identification of southern African snakes for use in the investigating and 1) Bitis albanica (Albany adder) Bitis arietans (Puff adder) 2) Bitis armata (Southern adder) Bitis atropos (Cape Berg adder) prosecution of illegal wildlife crime involving species; 3) Bitis gabonica (Gaboon adder) Bitis caudalis (Horned adder) 4) Bitis inornata (Plain mountain adder) Bitis cornuta (Many horned adder) Species selection: Priority listing Bitis rubida (Red adder) Species were selected for inclusion into the Barcode of Wildlife Project Bitis schneideri (Namaqua dwarf adder) Bitis xeropaga (Desert mountain adder) (BWP) databases based on their CITES listed and threatened species Bitis nasicornis (Rhinoceros viper) status in South Africa. The following are the four criteria used; 5) Dendroaspis polylepis (Black mamba) 6) D. angusticeps (Eastern green mamba) . Threatened species with small populations; traded in very high /high/ moderate/low volumes 7) Python natalensis (Southern African Python sebae (African rock python) python) Python anchietae (Angolan python) . Threatened species traded in very high, high to moderate volumes Python regius (Royal or Ball python) Python reticulatus (Reticulated python) Near Threatened , Data Deficient, Rare, Declining species traded in Boa constrictor (Boa constrictor) . 8) Others Naja annulifera (Banded cobra) high to very high volumes N. mossambica (Mozambique spitting cobra) Cape N. nivea (Cape cobra) . Species traded in very high volumes cobra N. nigricollis (Black-necked spitting cobra) N. melanoleuca (Forest cobra) Hemachatus haemachatus (Rinkhals) Philothamnus natalensis (Natal green snake) RESULTS P. semivariegatus (Spotted bush snake) . Twenty species of snakes from the families Boidae, Colubridae, Elapidae, Lamprophiidae, Viperidae were analysed using the COI barcoding gene. NZG234_Naja annulifera_Snouted cobra 68 NZG036_Naja annulifera_Snouted cobra 100 NZG235_Naja annulifera_Snouted cobra NZG034_Naja annulifera_Snouted cobra . A minimum of five samples per species were collected from zoos, 99 NZG035_Naja annulifera_Snouted cobra NZG276_Naja nivea_Cape cobra reserves and museum collections. 75 NZG454_Naja nivea_Cape cobra 100 NZG277_Naja nivea_Cape cobra NZG274_Naja mossambica_Mozambique spitting cobra NZG273_Naja mossambica_Mozambique spitting cobra 100 Cobras (4.2-15.6 %) . The COI genetic diversity estimates revealed high levels of sequence 96 NZG025_Naja mossambica_Mozambique spitting cobra 100 ISDB045-12|Naja naja|COI-5P BIN divergence between families (17–26 %) and among species within ISDB010-11|Naja naja|COI-5P 63 GBGCR285 Naja kaouthia BIN_U 99 BIN these groups (0.2-25 %). (Figure 1) GBGC12053-13|Naja atra|COI-5P|JN833604 100 BIN 100 GBGC7240-09|Naja atra|COI-5P|EU921898 NZG032_Naja melanoleuca_Forest cobra 86 NZG272_Naja melanoleuca_Forest cobra . There was also evidence of cryptic speciation and geographic variation 100 NZG271_Naja melanoleuca_Forest cobra 71 NZG033_Naja melanoleuca_Forest cobra among species from taxonomic groups such as the vipers and colubrids 82 NZG059_Hemachatus haemachatus_Rinkhals NZG060_Hemachatus haemachatus_Rinkhals with some species had more than one BIN number (Figure 1 & 2). 100 76 NZG057_Hemachatus haemachatus_Rinkhals NZG268_Dendroaspis angusticeps_Green mamba 100 NZG030_Dendroaspis polylepis_Black mamba 100 NZG278_Philothamnus semivariegatus_Spotted bush snake 97 NZG280_Philothamnus semivariegatus_Spotted bush snake NZG279_Philothamnus semivariegatus_Spotted bush snake 100 NZG504_Philothamnus semivariegatus_Spotted bush snake Colubridae - Cryptic? South African rock python NZG037_Pseudaspis cana_Mole snake2 NZG039_Pseudaspis cana_Mole snake 100 100 NZG856_Pseudaspis cana_Mole Snake NZG450_Boa constrictor_Boa constrictor Boidae? Pythons > 21% 100 NZG451_Boa constrictor_Boa constrictor 72 NZG042_Bitis arietans_Puff adder 83 NZG012_Bitis arietans_Puff adder 100 New? 5.3-7.1 (0.2-1.6) % NZG041_Bitis arietans_Puff adder 100 NZG236_Bitis arietans_Puff adder 100 EANAH424-12|Bitis arietans BIN1 BIN GBGC7713-09|Bitis arietans BIN2 BIN 78 Vipers (4.6-25 %) 100 NZG501_Bitis nasicornis_Rhinoceros viper 93 NZG502_Bitis nasicornis_Rhinoceros viper 100 NZG338_Bitis gabonica_Gaboon viper 100 NZG352_Bitis gabonica_Gaboon adder NZG040_Bitis gabonica_Gaboon adder Spotted bush snake 98 verify >7 (0.4) % 100 NZG455_Bitis nasicornis_Rhinoceros viper 91 NZG045_Python natalensis_South African python 100 NZG044_Python natalensis_South African python NZG275_Python natalensis_Southern African python NZG457_Python reticulatus_Reticulated python 96 Boidae - Pythons (10-17 %) NZG043_Python regius_African ball python 51 NZG507_Python regius_Ball python 100 NZG505_Python regius_Ball python 78 67 NZG506_Python regius_Ball python NZG351_Alligator mississippiensis_Alligator 100 NZG350_Alligator mississippiensis_Alligator Figure 1: DNA100 barcoding (COI) tree NZG349_Alligator mississippiensis_Alligator AF511507 Alligator sinensis neighbor joining tree100 NZG344_Mecistops of southern cataphractus_African African slender snouted crocodile Crocs 99 NZG347_Mecistops cataphractus_African slender snouted crocodile snake taxa NZG342_Osteolaemus tetraspis_African dwarf crocodile Puff adder 100 NZG341_Osteolaemus tetraspis_African dwarf crocodile 100 NZG340_Osteolaemus tetraspis_African dwarf crocodile JQ909470 Madascincus melanopleura Skink Lizards (Map:99 Distribution NZG281_Cordylus of the cataphractus_Armadillo puff adder lizard in Africa) Gaboon viper (S. Africa) Rhino viper SIGNIFICANCE . This database indicated that DNA barcoding will be useful for snake species identification in southern Africa. The observed diversity levels further indicate that some snake families may be in need of taxonomic revaluation as there were very high levels of intraspecific variation. Figure 2: Photographs of reference samples used in the BWP data set showing examples of morphological variation within . This DNA barcoding information will improve our current and among species (e.g. vipers, spotted bush snakes and rock understanding of snake communities that can inform more pythons from South Africa). accurate predictions of future impacts of global change. Funding Sources/Acknowledgements 1. Marli De Bruyn & Thando Radebe (NZG) References 2. Dr Graham Alexander – WITS 1. Alexander G. & Marais J. 2007. A guide to the reptiles of southern Africa. Random House 3. http://www.sareptiles.co.za (photos) Struik (Pty) Ltd, South Africa, pp 1-408. 4. D. Schindel, M. Trizna, M. Parker & N. Redmond 5. SANBI (RSA Project Coordination) – M. Hamer 6. SAPS (FSL), DEA & NPA – Implementing agencies Email: [email protected].
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